Synthesis of the Phosphoramidite Derivative of 2′-Deoxy-2′-C-β-methylcytidine
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Nan-Sheng Li and Joseph A. Piccirilli* Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology and Department of Chemistry, University of Chicago, 5841 South Maryland Avenue, MC 1028, Chicago, Illinois 60637
[email protected] Received February 27, 2003
Abstract: 2′-Deoxy-2′-C-β-methylnucleosides elicit interest as potential therapeutic agents and as analogues for the analysis of nucleic acid structure and function. An efficient route for the synthesis of 2′-deoxy-2′-C-methyluridine (11), 2′-deoxy-2′-C-methylcytidine (12), and the phosphoramidite derivative of 2′-deoxy-2′-C-β-methylcytidine (10, 46% overall yield) from 1,2,3,5-tetra-O-benzoyl-2-C-β-methylribofuranose (1) is described.
The 2′-hydroxyl group plays an integral role in RNA structure and function, mediating hydrogen bonds, coordinating to metal ions, and providing a scaffold for interactions with water.1 The locations of residues bearing important 2′-hydroxyl groups therefore provide fundamental clues about the structure and function of an RNA. Deoxynucleotide substitution provides the most common approach by which to locate these residues, as the removal of an important 2′-hydroxyl group has deleterious consequences for RNA function.2 However, diminished activity from deoxynucleotide substitution cannot be interpreted monolithically as evidence for interactions with the 2′-hydroxyl group because the loss of RNA function could be due to an altered conformational preference of the deoxynucleotide. Ribonucleotides exhibit a preference for the 3′-endo sugar conformation, but deoxyribonucleotides lack this preference, populating the 2′- and 3′-endo conformations equally well (Scheme 1).3 A molecular framework that allows removal of the 2′-hydroxyl group without a concomitant change in sugar pucker would eliminate this conformational ambiguity and thereby clarify the interpretation of deoxynucleotide substitution experiments. We suggest that 2′-β-methylnucleotides offer such a context as they maintain the 3′endo conformational preference even in the absence of the 2′-hydroxyl group (Scheme 1).4 To conduct 2′-deoxynucleotide substitution experiments with RNA using 2′-β-methylnucleotides, we must (1) Auffinger, P.; Westhof, E. J. Mol. Biol. 1997, 274, 54 and references therein. (2) Gesteland, R. F.; Cech, T. R.; Atkins, J. F. The RNA World, Second Edition; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 1999. (3) Saenger, W. Principles of Nucleic Acid structure; SpringerVerlag: Berlin, Germany, 1983. (4) (a) Schmit, C.; Bevierre, M.-O.; Mesmaeker, A. D.; Altmann, K.H. Bioorg. Med. Chem. Lett. 1994, 4, 1969. (b) Cicero, D. O.; Iribarren, A. M.; Bazzo, R. Appl. Magn. Reson. 1994, 7, 95.
develop the syntheses of the ribo and deoxynucleosides and their phosphoramidites. Here we report an efficient synthesis of 2′-deoxy-2′-C-β-methylcytidine and the corresponding phosphoramidite for incorporation into oligoribonucleotides. We chose cytidine for its application to our mechanistic work on the group II intron ribozyme5 and because 2′-deoxy-2′-C-β-methylcytidine exhibits potent cytotoxicity toward L1210 cells.6 Two approaches have been described to access 2′-deoxy2′-methylnucleosides. Cicero et al.7 and Schmit et al.4a used catalytic hydrogenation of a suitably protected 2′methylene nucleoside.8 The reduction proceeds quantitatively but suffers from relatively weak diastereoselectivity (β/R ) 3:1; diastereomeric excess 50%). Cicero et al. subsequently used this mixture as the starting material to prepare the phosphoramidite derivative of 2′deoxy-2′-C-β-methyl-N4-isobutyrylcytidine in six steps with 20% overall yield.9 In the other approach, Matsuda et al. obtained 2′-deoxy-2′-C-β-methylcytidine from uridine in 10 steps via synthesis of intermediate I followed by 2′-deoxygenation, sequential deblocking and amination.6 This approach has limited efficiency (9% overall yield from uridine) because I forms as a minor product from the Grignard reaction of the corresponding 2′ketonucleoside with methylmagnesium bromide.10
After the reports of Cicero and Matsuda, O’kuru established a facile synthesis of 2′-C-β-methylribonucleo(5) Gordon, P. M.; Sontheimer, E. J.; Piccirilli, J. A. Biochemistry 2000, 39, 12939. (6) Matsuda, A.; Takenuki, K.; Sasaki, T.; Ueda, T. J. Med. Chem. 1991, 34, 234. (7) Cicero, D. O.; Neuner, P. J. S.; Franzese, O.; D’Onofrio, C.; Iribarren, A. M. Bioorg. Med. Chem. Lett. 1994, 4, 861. (8) Samano, V.; Robins, M. J. Synthesis 1991, 283. (9) Cicero, D. O.; Gallo, M.; Neuner, P. J.; Iribarren, A. M. Tetrahedron 2001, 57, 7613.
10.1021/jo034263y CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/22/2003
J. Org. Chem. 2003, 68, 6799-6802
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SCHEME 2a
SCHEME 3a
a Key: (i) TsCl, K CO /CH CN, 85 °C, 2 h; (ii) 2 N NaOEt/EtOH, 2 3 3 reflux, 1 h; (iii) TIPDSCl2, imidazole, DMF, rt, overnight; (iv) SOCl2/CHCl3/DMF, reflux, 6.5 h.
sides via nucleobase glycosylation with suitably protected 2-β-methylribofuranose.11 We anticipated that this method could help to improve the synthesis of 2′-deoxy-2′-C-βmethylnucleosides by facilitating access to 2′-C-β-methylribonucleosides, which may serve as precursors either for conversion to I or for direct 2′-deoxygenation. First, we explored the synthesis of intermediate I by transformation of 2′,3′,5′-tri-O-benzoyl-2′-C-β-methyluridine (2). O’kuru obtained 2 with complete stereoselectivity in 57% yield by reaction of 1,2,3,5-tetra-O-benzoyl-2-C-β-methylribofuranose (1) with bis(trimethylsilyl)uracil, prepared in situ from N,O-bis(trimethylsilyl)acetamide and uracil.11 We used hexamethyldisilazane12,13 instead of N,O-bis(trimethylsilyl)acetamide and improved the yield of 2 substantially (96%). However, attempts to transform 2 into intermediate I gave low yields following reaction sequences using either thionyl chloride10 or tosyl chloride14 (Scheme 2). We then investigated whether suitably protected derivatives of 2′-C-β-methylcytidine or 2′-C-β-methyluridine would undergo 2′-deoxygenation directly to give the corresponding 2′-deoxynucleoside. We prepared 2′-C-βmethyl-N4-benzoylcytidine and 2′-C-β-methyluridine as described11,12 and protected them as 3′,5′-O-1,1,3,3-tetraisopropyldisiloxyl ethers. The cytidine derivative failed to undergo 2′-deoxygenation via either the phenoxythiocarbonyl15 or methyl oxalyl ester6 using Bu3SnH in the presence of AIBN. However, deoxygenation of the corresponding uridine analogue proceeded efficiently and stereoselectively (β/R ) 93:7; diastereomeric excess 86%), thereby enabling facile access to 2′-deoxy-2′-C-β-methyluridine, 2′-deoxy-2′-C-β-methylcytidine and the phosphoramidite of 2′-deoxy-2′-C-β-methylcytidine in high yield and with greater diastereoselectivity than reported previously. Scheme 3 shows our overall synthesis of the phosphoramidite derivative of 2′-deoxy-2′-C-β-methylcytidine. Treatment of 2 with saturated ammonia in methanol gave 2′-C-β-methyluridine (3) quantitatively. Protection of the 3′- and 5′-hydroxyl groups of 3 using 1,3-dichloro1,1,3,3-tetraisopropyldisilane gave 3′,5′-tetraisopropyldisiloxan-1,3-diyl-2′-C-β-methyluridine (4) in 65% yield. (10) Matsuda, A.; Itoh, H.; Takenuki, K.; Sasaki, T.; Ueda, T. Chem. Pharm. Bull. 1988, 36, 945. (11) Harry-O’kuru, R. E.; Smith, J. M.; Wolfe, M. S. J. Org. Chem. 1997, 62, 1754. (12) Tang, X.-Q.; Liao, X.-M.; Piccirilli, J. A. J. Org. Chem. 1999, 64, 747. (13) Li, N.-S.; Tang, X.-Q.; Piccirilli, J. A. Org. Lett. 2001, 3, 1025. (14) Czernecki, S.; Mulard, L.; Valery, J.-M.; Commercon, A. Can. J. Chem. 1993, 71, 413. (15) Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem. Soc. 1983, 105, 4059.
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a Key: (i) bis(trimethylsilyl)uracil, SnCl , CH CN, rt, 20 h; (ii) 4 3 NH3, CH3OH, 0-4 °C, 2 days; (iii) TIPDSCl2, imidazole, DMF, rt, 2 h; (iv) ClCOCO2Me, DMAP, CH3CN, rt, 1 h; (v) n-Bu3SnH, AIBN, toluene, reflux, 2 h; (vi) TPSCl, DMAP, Et3N, CH3CN, rt, 53 h; (vii) NH4OH, rt, 3 h; (viii) BzCl, DMAP, CH2Cl2, rt, 1 h; (ix) Et3N/ 3HF, THF, rt, 4 h; (x) DMTrCl, pyridine, rt, overnight; (xi) CIP (NPr-i2)OCH2CH2CN, i-Pr2NEt, 1-methylimidazole, CH2Cl2, rt, 2 h.
Deoxygenation of 4 via free-radical chemistry gave 3′,5′tetraisopropyldisiloxan-1,3-diyl-2′-deoxy-2′-C-β-methyluridine (5) in 97% yield with β/R selectivity of 93:7 (diastereomeric excess 86%). Reaction of 5 with 2,4,6triisopropylbenzenesulfonyl chloride16 followed by treatment with ammonium hydroxide gave the corresponding cytidine derivative (6) in 91% yield. Benzoylation of 6 generated 7 in 93% yield and enabled complete removal of the R-isomer by silica gel column chromatography. Removal of the silyl group from 7 with triethylamine trihydrofluoride gave 2′-deoxy-2′-C-β-methyl-N4-benzoylcytidine (8) quantitatively. Compound 8 was then converted to the phosphoramidite derivative of 2′-deoxy-2′C-β-methylcytidine (10) in 90% yield. Deprotection of compounds 5 and 6 gave the free nucleosides 2′-deoxy-2′-C-methyluridine (11) and 2′-deoxy2′-C-methylcytidine (12) in high yields as diastereomeric mixtures (β/R ∼93:7). We were unable to separate the diastereomers by silica gel chromatography using as eluent either 10% methanol in ethyl acetate or 20% methanol in chloroform (Scheme 4). We confirmed by (16) Iino, T.; Yoshimura, Y.; Matsuda, A. Tetrahedron 1994, 50, 10397.
SCHEME 4
NOESY NMR spectra of 11 that the major isomer has the 2′-β-configuration. We observed a strong NOE between 1′-H (δ 6.20) and 2′-H (δ 2.52), weaker NOE’s from 1′-H (δ 6.20) to 2′-C-Me (δ 0.95) and 4′-H (δ 3.72), and no NOE from 1′-H (δ 6.20) to either 3′-H (δ 3.88) or 5′-H (δ 3.78, 3.93). These results suggest that 1′-H and 2′-H reside on the same face of the ribose ring. We also observed a strong NOE between 6-H (δ 8.08) and 3′-H (δ 3.88) and a weaker NOE between 6-H (δ 8.08) and 2′-CMe (δ 0.95), consistent with the proposed preference of 2′-deoxy-2′-C-β-methylnucleosides for the 3′-endo conformation.4 In conclusion, 2′-C-β-methyl-2′-deoxynuclesides may provide a means to eliminate the conformational ambiguity inherent to 2′-deoxynucleoside substitution experiments in functional RNA molecules. As part of our efforts to test this hypothesis, we have established more facile access to the phosphoramidite derivative of 2′-deoxy-2′C-β-methylcytidine from a sugar substrate: 1,2,3,5-tetraO-benzoyl-2-C-β-methylribofuranose. This approach has greater stereoselectivity and a higher overall yield than previously published methods. NOESY experiments support previous arguments based on NMR coupling constants4 that 2′-deoxy-2′-C-β-methylnucleosides populate the 3′-endo conformation. Experimental Section 2′,3′,5′-Tri-O-benzoyl-2′-C-β-methyluridine (2). Under an argon atmosphere, a stirred suspension of uracil (1.12 g, 10.0 mmol) and (NH4)2SO4 (25 mg) in 1,1,1,3,3,3-hexamethyldisilazane (25 mL) was heated at reflux until a clear solution formed. The clear solution was concentrated under vacuum, and the residue was dried under high vacuum (
